Much attention has focused on methods for generating immune reactions. One class of immune reaction to foreign antigens is the production of antibodies, typically referred to as humoral immunity. A second form of immune reaction results from the presentation of antigen by an antigen presenting cell (APC). This type of immune reaction is broadly referred to as cell mediated immunity (CMI), or T cell responses. Although both types of immune responses are important, considerable attention has recently focused on CMI. In dealing with infectious diseases such as AIDS, caused by the Human Immunodeficiency Virus (HIV), the antibody responses to the virus and portions thereof have not proven sufficient to confer immunity. Similarly, in dealing with exogenous proteins associated with many malignancies, the antibody responses have also not proven sufficient. Thus, speculation has focused on generating CMI responses.
In order to elicit CMI, an antigen must be bound to a major histocompatibility complex (MHC) class I or II molecule on the surface of the APC. The class I molecules typically present antigens externally, such as endogenous proteins, those from viral infections, and tumor antigens. Antigen-specific T cells typically recognize infected target cells when the pathogen-derived (or cancerous) peptide epitopes (usually 8 to 10 amino acids) are presented by molecules encoded by the host class I MHC (7). These epitopes are derived from cytoplasmic proteins cleaved by the proteosome into small peptide fragments. These are then transported into the lumen of the endoplasmic reticulum (ER), where they complex with newly synthesized MHC-I molecules and are subsequently transported to the cell surface, where recognition by T cells occurs (8-13). Antigens in the extracellular fluid (exogenous antigens) generally do not gain access into this processing compartment in most cells. Thus, a significant challenge to eliciting CMI with a vaccine is the delivery of exogenous antigens to the cytosol for presentation by MHC class I molecules. It would be desirable to be able to generate vaccines to a wide variety of infectious diseases, such as HIV, as well as cancers, such as prostate cancer, breast cancer and melanoma.
For example, growing evidence suggests that CMI plays an essential role in controlling HIV infection (Ogg et al., Science 279:2103-6 (1998); Schmitz et al., Science 283:857-60 (1999); Brodie et al., Nat. Med. 5:34-41 (1999)). Individuals who have been exposed to HIV but remain uninfected often have antiviral CMI but no antibody response. The viremia of primary infection resolves as viral specific cytotoxic T lymphocytes (CTL) develop, before the development of specific antibodies (Letvin, Science 280:1875-96 (1998)). These data illustrate the central role CMI plays in controlling HIV infection.
Many tumors are associated with the expression of a particular protein and/or the over-expression of certain proteins. For example, prostate cancer is associated with elevated levels of protein such as Prostate Specific Antigen (PSA). Breast cancers can be associated with the expression and/or over-expression of protein such as Her-2, Muc-1, CEA, etc. Thus, considerable attention has been aimed at trying to generate immune responses, particularly developing CMI, to such antigens in the treatment of such malignancies.
Approaches to developing cell mediated immunity to infectious diseases have included using the entire infectious agent, for example, by making genetically engineered inactivated viruses or using a killed infectious agent. Another approach has been subunit vaccines, which is presenting one or more antigens (but not the entire virus) to a subject.
In order to generate CMI, antigen must be delivered to the interior of the cell. Exogenous proteins are poorly taken up by the cell. Accordingly, the preferred method has been using procedures such as viral vectors, liposomes, naked DNA or a similar approach. However, such approaches have many draw backs. For example, many recombinant viruses generate antigenic reactions themselves, upon repeated administration. Since standard forms of generating immune reactions typically require an initial injection, referred to as the prime, and subsequent injections, referred to as boosts, to achieve a satisfactory immunity, this can be a serious problem. Moreover, while much attention has been placed on improving the safety of viral vectors, there are always certain risks. For example, many of the target populations, such as those infected with HIV, may have a weakened immune system. Thus, certain viral vectors that are perfectly safe in many individuals may pose some degree of risk to these individuals. Methods of delivering protein to cells have also not proven entirely satisfactory as of this time. Accordingly, there is a need for new and simple methods to deliver an antigen to the cytosol to stimulate CMI.
In trying to develop CMI responses, there have also been technical problems with the difficulty in measuring these responses.
Current available laboratory assays to detect cell-mediated immune responses have serious shortcomings, especially when applied to large vaccine efficacy trials in various clinical settings. This is because the available equipment and technical support required to measure CMI using current techniques are often minimal in the setting where they are required, in the field. CTL are thought to play a crucial role in controlling HIV-1 infection, and many HIV-1 vaccine candidates are designed to stimulate T cell responses as well as neutralizing antibodies (1-6). However, the standard laboratory methods for detecting CMI, such as HIV-specific CTL, are complex, time consuming, and often restricted to highly specialized facilities. An improved method for measuring T cell responses will have a significant effect on the development of all T cell dependent vaccines or immune therapies. This strategy can potentially be applicable to other fields of research where CMI responses are known to play an important role in prevention and control of the diseases.
One difficulty in reliably detecting CMI response in vitro results from the unique requirement for antigen presentation. As described above, the delivery of exogenous proteins to the cytosol for presentation to T cells by MHC class I molecules represents a significant challenge. This physical partition of the class I pathway has been a major barrier to detect T cell responses in vitro. Consequently, most of the current laboratory methods in measuring CMI utilize live viral or bacterial vectors to deliver antigens into cytosol, among which recombinant pox such as vaccinia viruses are the most commonly used. Another approach is to externally load MHC-I molecules on surface of target cells with synthetic peptides (10 to 20 amino acids) derived from known CTL epitopes. These methods have serious limitations in general clinical uses. The use of a live viral vector, such as a recombinant vaccinia virus, requires trained and immunized laboratory personnel, including minimum containment facilities as precautionary safety measures. Synthetic peptides are not only prohibitively expensive, but the design of the “universal” peptide profile that fit the diversified MHC-I molecules in various populations is extremely difficult. The challenge for the development of assays to measure T cell responses is, therefore, to deliver large pieces of exogenous antigens into cytosol without resorting to live recombinant viral or bacterial vectors.
Accordingly, it would be desirable to have kits that could be used for measuring CMI in vitro. It would be particularly desirable to have kits that could be readily used in remote locations such as Africa, India and Asia, where there are many proposals to test a number of vaccine candidates such as vaccines against HIV.
We have now discovered that a family of bipartite protein exotoxins, such as Bacillus anthracis, contains fragments that can be used for the delivery of exogenous antigens, such as proteins, to the cytosol. One preferred protein fragment from these proteins is from the N-terminal portion that contains the protective antigen (PA) binding domain, but not those portions resulting in toxicity to the cell. More preferably, that fragment has been modified to remove the specific domain that binds to PA.
B. anthracis is the causative agent of anthrax in animals and humans. The toxin produced by B. anthracis consists of two bipartite protein exotoxins, lethal toxin (LT) and edema toxin. LT is composed of protective antigen (PA) and lethal factor (LF), whereas edema toxin consists of PA and edema factor (EF). None of these three components, PA, LF, and EF, alone is toxic. Once combined however, edema toxin causes edema and LT causes death by systemic shock in animals and humans. Consistent with its critical role in forming both toxins, PA has been identified as the protective component in vaccines against anthrax. The molecular mechanism of anthrax toxin action is currently hypothesized as follows: PA is a 735-amino acid polypeptide that binds to the surface of mammalian cells by cellular receptors. Once bound, PA is activated by proteolytic cleavage by cellular proteases to a 63-kDa molecule capable of forming a ring-shaped heptamer in the plasma membrane of the targeted cell (FIG. 1) (6, 7). The PA heptamer then binds either EF or LF, which are internalized by endocytosis. After endosomal acidification, PA enables EF or LF to enter the cytosol, presumably by means of a pore formed by the heptamer. Within the cytosol, EF acts as an adenylate cyclase (8) to convert ATP to cAMP. Abnormally elevated levels of cAMP perturb cellular metabolism.
The action of LF in the cytosol causes the death of host cells by a mechanism that is not well understood. LF induces over-production of a number of lymphokines (9), contributing to lethal systemic shock in host animals. Recent studies also show that LF has two enzymatic activities: it can act as a zinc metalloprotease (10), and it inactivates the mitogen-activated protein kinase (11). Although it is still not clear how these two enzymatic activities of LF are connected, both are required for LF toxicity. It has previously been reported that anthrax toxin B moieties may be used to deliver eptiopes which in turn elicit an antibody response by the immune system, in the presence of PA (WO 97/23236).
LF is a 776 aa polypeptide, and the functional domain for both enzymatic activities is located between amino acids 383 and 776 of LF (FIG. 2A; SEQ ID NO: 1). The N-terminal truncated LF (LFn) polypeptide is a 255 amino acid polypeptide (corresponding to residues 34-288 of SEQ ID NO: 2). The 255 amino acid LFn polypeptide part is derived from a precursor protein of 1-288 residues as shown in FIG. 2B (SEQ ID NO:2), where the first 1-33 amino acids correspond to the signal peptide. Without the catalytic domain, LFn polypeptide (residues 34-288 of SEQ ID NO: 2) completely lacks any toxic effect when mixed with PA and added to cultured macrophages or when injected into animals. It does, however, still bind to PA effectively. The PA binding domain of the LFn polypeptide is located within the first 1-149 N-terminal amino acids of the LFn polypeptide (i.e. where the LFn polypeptide is residues 34-288 of SEQ ID NO: 2, thus the first 1-149 N-terminal amino acids of the LFn polypeptide are residues 34-184 of SEQ ID NO:2.